Trial Information

Randomized Phase II Study of Immunization With MAGE-3/Melan-A/gp 100/NA17 Peptide-Pulsed Autologous PBMC and rhIL-12 With or Without Low Dose IL-2 Inpatients With Metastatic Melanoma

Based on the above preclinical and Phase I results, a logical strategy for a second
generation melanoma vaccine has emerged. A randomized Phase II study in metastatic melanoma
patients will be undertaken. Patients first will be HLA-typed; HLA-A2-positive patients will
be eligible for screening. When feasible, each patient will undergo a tumor biopsy to screen
for expression of MAGE-3, Melan-A, gplOO, and NAI 7 using RT-PCR and immunohistochemistry,
to determine whether T cells are present in the lesion, to measure cytokine gene expression
by RT-PCR, and to perform gene array analysis. In addition, blood cells will be analyzed for
certain parameters of T cell function.

Patients will be randomized to cohorts A (no IL-2) or B (with low-dose IL-2). For treatment,
peripheral blood will be collected and fractionated by density centrifugation to isolate
PBMC as a source of APC. The PBMC will be divided into four pools, each of which will be
incubated with one of the following peptides: MAGE-3, Melan-A, gp 100, or Ni 7A. The
peptide-loaded cells will then be washed and recombined into a single suspension in PBS, and
lethally irradiated. Approximately 120 x 106 pulsed cells will be injected subcutaneously at
a site near a lymph node not thought to be involved with tumor. The subcutaneous route has
been selected for the reasons of safety, efficacy in the preclinical model, and the goal of
targeting the vaccine to a draining lymph node. rhIL-12 (4 .tg straight dose) will then be
given subcutaneously adjacent to the vaccine site days 1,3, and 5 of each cycle. This dose
and schedule was found to be effective in our phase I study. In one-half of the patients
(cohort B), IL-2 (I MU straight dose) will be administered subcutaneously daily, days 7-18.
Re-immunization along with rhIL-12 followed by IL-2 (if assigned) will be performed at 3
week intervals as in cycle I.

On day 1 of each cycle, peripheral blood will be collected to measure peptide-specific IFN-y
production. Before treatment and after every 3 cycles, PBMC will be collected to quantify
peptide specific CD8 T cells by flow cytometric analysis with peptide/HLA-A2 tetramers, and
evidence for a molecular response will be assessed by performing RT-PCR. for melanoma
antigens on peripheral blood samples. In addition, prior to treatment, after the first 3
cycles, and at the time of going off- study, a tumor biopsy will be performed to assess the
immune response in the tumor microenvironment, including gene array analysis. It is hoped
that these studies will uncover the reason for lack of clinical response in patients with
residual tumors. Clinical response will be assessed as a secondary outcome.